Plants are highly flexible organisms, forced to efficiently and quickly adapt to (changes in) their environment. Unable to move, they have evolved morphological and physiological strategies that allow growth even in challenging environments. However, environmental adaptation is not always in harmony with optimal economic traits desired by the farmer or the consumer. Plants, which are fully adapted to a specific environment often have relatively low yields or nutritional value or lack ornamental characteristics. Conversely, heavily bred varieties designed to fit the needs of farmers and consumers, are often affected by environmental circumstances and/or changes.
Abiotic stress or environmental stress is stress caused to plants in other ways than through living organisms. Examples of abiotic stress are environmental conditions such as: high salinity, osmotic stress, oxidative stress, (extreme) heat and (extreme) cold and drought. Crop losses and crop yield losses of major crops such as rice, maize (corn) and wheat caused by these stresses represent a significant economic and political factor and contribute to food shortages in many developing countries.
Plants are typically exposed during their life cycle to conditions of reduced environmental water content. Most plants have evolved strategies to protect themselves against these conditions of desiccation. However, if the severity and duration of the drought conditions are too great, the effects on plant development, growth and yield of most crop plants are profound. Furthermore, most of the crop plants are very susceptible to higher salt concentrations in the soil. Continuous exposure to drought and high salt causes major alterations in the plant metabolism. Similar alterations can be observed by prolonged exposure to extreme heat or cold. These great changes in metabolism ultimately lead to cell death and consequently yield losses.
In 1979 a novel plant growth-promoting factor, termed brassinolide, was isolated from the pollen of rape (Brassica napus) and identified as a novel type of steroid lactone. It was found that brassinolide-like steroid compounds (called brassinosteroids) occur in all plant species examined at very low concentrations and had a function in adapting the plants to combat both biotic and abiotic stress (for review, see Mandava, Ann. Rev. Plant Physiol. Plant Mol. Biol. 39 (1988), 23-52). Initial studies of the physiological action of brassinolide showed that this particular factor (i) accelerated the germination and growth of plant seedlings at low temperatures, (ii) promoted the increase of cell size and elongation by induction of a longitudinal arrangement of cortical microtubuli and cellulose microfilaments on the surface of cells, (iii) promoted xylem differentiation by amplifying the tracheal elements, (iv) resulted in significant increase of dry weight of plants and their fruits, (v) promoted leaf unrolling and enlargement, (vi) induced H+ export and membrane hyperpolarization characteristic for auxin induced cell growth, (vii) inhibited the division of crown-gall tumour cells and radial growth of stems, (viii) repressed anthocyanin production in light-grown plants, (ix) inhibited the de-etiolation induced, e.g. by cytokinin in the dark, (x) promoted tissue senescence in the dark, but prolonged the life-span of plants in the light and (xi) induced plant pathogen resistance responses to numerous bacterial and fungal species (listed by Mandava (1988), loc. cit.). Recent work has further confirmed the protective role of brassinosteroids against a wide range of abiotic stresses (drought, cold and salt, Kagale et al., Planta 225 (2007), 353-364).
Following the initial isolation of and physiological studies with brassinolides, numerous brassinosteroid compounds, representing putative biosynthetic intermediates, were identified in different plant species. Because the in vivo concentration of these compounds was found to be extremely low, efforts had been made to develop methods for chemical synthesis of these compounds (for review, see: Adam and Marquardt, Phytochem. 25 (1986), 1787-1799). These compounds were tested in field experiments using soybean, maize, rice and other crops as well as trees in order to confirm the results of physiological studies. However, the field trials showed that due to poor uptake of steroids through the plant epidermis, the amount of steroids required for spraying or fertilization was considerable, thereby making the use of brassinosteroids for providing plants with resistance to (a) biotic stress practically impossible.
Developing stress-tolerant plants is a strategy that has the potential to solve or mediate at least some of these problems. However, traditional plant breeding strategies to develop new lines of plants that exhibit resistance (tolerance) to these types of stresses are relatively slow and require specific resistant lines for crossing with the desired line. Limited germplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species represent significant problems encountered in conventional breeding. Additionally, the cellular processes leading to drought, heat/cold, salt and other tolerances in model tolerant plants are complex in nature and involve multiple mechanisms of cellular adaptation and numerous metabolic pathways. This multi-component nature of stress tolerance has not only made breeding for tolerance largely unsuccessful, but has also limited the ability to genetically engineer stress tolerance plants using biotechnological methods.
Therefore, what is needed is the identification of the genes and proteins involved in these multi-component processes leading to stress tolerance. Elucidating the function of genes expressed in stress tolerant plants will not only advance our understanding of plant adaptation and tolerance to environmental stresses, but also may provide important information for designing new strategies for crop improvement.
Expression and function of abiotic stress-inducible genes have been well studied at a molecular level. Complex mechanisms seem to be involved in gene expression and signal transduction in response to the stress. These include the sensing mechanisms of abiotic stress, modulation of the stress signals to cellular signals, translocation to the nucleus, second messengers involved in the stress signal transduction, transcriptional control of stress-inducible genes and the function and cooperation of stress-inducible genes.
In animal cells, phosphatidylinositol-specific phospholipase C (PI-PLC) plays a key role in early stages of various signal-transduction pathways. Extracellular stimuli such as hormones and growth factors activate PI-PLCs. PI-PLC hydrolyzes phosphatidylinositol 4,5-biphosptate (PIP2) and generates two second messengers, inositol, 4,5-triphosphtate (IP3) and 1,2-diacylglycerol (DG). IP3 induces the release of intracellular Ca<2+> into the cytoplasm, which in turn causes various responses therein. DG and PIP2 also function as second messengers and control various cellular responses.
In plants, similar systems are thought to function in abiotic stress response. It is clearly demonstrated that phospholipases A, C or D (PLA, PLC or PLD), depending upon their site of cleavage, play a role in the early signal transduction events that promote the cell volume changes associated with osmotic stress and osmoregulation in plants which is important for plant stress tolerance (Wang X. et at., 2000, Biochemical Society Transactions. 28; 813-816; Chapman K D, 1998 Tr. Plant Sci. 3:419-426). For example, in guard cells, abscisic acid (ABA)-induced stomatal closure is mediated by rapid activation of PIP2-PLC. This leads to an increase in IP3 levels, a rise in cytosolic calcium, and the subsequent inhibition of K+ channels. For example, a gene for phospholipase C, AtPLC was demonstrated to be rapidly induced by drought and salt stresses in Arabidopsis thaliana (Hirayama, T. et al., 1995 Proc. Natl. Acad. Sci. 92:3903-3907).
As mentioned above, Ca2+ ions play important roles as second messengers in various signal-transduction pathways in plants. Marked increase in intracellular Ca2+ concentration has been observed upon stimulation by wind, touch, abiotic stresses (cold, drought and salinity) or fungal elicitors. Several genes for Ca2+ binding proteins with a conserved EF-hand domain have been isolated and showed increased expression level upon abiotic stress treatment (Frandsen G. et al., 1996 J. Biol. Chem. 271:343-348; Takahashi S. et al., 2000 Pant Cell Physiol. 41:898-903).
The enigmatically named 14-3-3 proteins have been also the subject of considerable attention in recent years since they have been implicated in the regulation of diverse physiological processes in eukaryotes ranging from slime moulds to higher plants. In plants, many biological roles for 14-3-3 proteins have been suggested. The most significant of these include roles in the import of nuclear encoded chloroplast proteins, in the assembly of transcription factor complexes and in the regulation of enzyme activity in response to intracellular signal transduction cascades (Chung H J. et al., 1999 Tr. Plant Sci. 4:367-371). The native 14-3-3 proteins are homo- or heterodimers and, as each monomer has a binding site, a dimer can potentially bind two targets, promoting their association. Alternatively, target proteins may have more than one 14-3-3-binding site.
Several functions have been proposed for the 14-3-3 proteins in terms of involvement of plant stress tolerance. The 14-3-3 proteins could function as regulators in stress signal transduction. For example, RCI14A and RCI14B genes are induced by cold treatment in Arabidopsis and are highly homologous to the 14-3-3 proteins. The rise in the RCI transcript levels observed in response to cold treatment suggests a role for the RCI proteins in the stress signalling transduction pathway (Jarillo J A et al., 1994 Plant Mol. Biol. 25:693-704)
Due to the commercial consequences of environmental damage to crops, there is an interest in understanding the stress response signal transduction mechanisms in plants and how these can be manipulated to improve a plant's response to environmental damage. There is a need, therefore, to identify genes expressed in stress tolerant plants that have the capacity to confer stress resistance to its host plant and to other plant species. Newly generated stress tolerant plants will have many advantages, such as increasing the range that crop plants can be cultivated by, for example, decreasing the water requirements of a plant species.